Hierarchical assembly of b2-microglobulin amyloid in vitro revealed by atomic force microscopy

The kinetics of spontaneous assembly of amyloid fibrils of wild-type b2-microglobulin (b2M) in vitro, under acid conditions (pH 2.5) and low ionic strength, has been followed using thioflavin-T (ThT) binding. In par-allel experiments, the morphology of the different fibrillar species present at different time-points during the growth process were characterised using tapping-mode atomic force microscopy (TM-AFM) in air and nega-tive stain electron microscopy (EM). The thioflavin-T assay shows a characteristic lag phase during which the nucleation of fibrils occurs before a rapid growth in fibril density. The volume of fibrils deposited on mica measured from TM-AFM images at each time-point correlates well with the fluorescence data. TM-AFM and negative-stain EM revealed the presence of various kinds of protein aggregates in the lag phase that dis-appear concomitantly with a rise in the density of amyloid fibrils, suggesting that these aggregates precede fibril growth and may act as nucleation sites. Three distinct morphologies of mature amyloid fibrils were observed within a single growth experiment, as observed previously for the wild-type protein and the variant N17D. Additional supercoiled morphologies of the lower-order fibrils were observed. Comparative height analysis from the TM-AFM data allows each of the mature fibril types and single protofilaments to be identified unambiguously, and reveals that the assembly occurs via a hierarchy of morphological states